It is well established that stimuli associated with drugs can be powerful modulators of drug-seeking behavior. While there have been a great deal of studies examining environmental/contextual stimuli, interoceptive stimuli have received significantly less attention. The current experiments combine a highly effective behavioral training procedure with cutting-edge viral technology in order to assess the role of the medial prefrontal cortex (mPFC) in combined nicotine+alcohol (N+A) use/abuse. In doing so, we will incorporate immunohistochemical assays to explore changes in projections to the nucleus accumbens core (AcbC), a structure known to be important in several aspects of drug abuse. For these experiments, rats will be trained to discriminate a compound N+A interoceptive cue from water. In order to assess the role of mPFC, rats will receive bilateral virus infusions of Cre dependent Gi-coupled hM4Di Designer Receptors Exclusively Activated by Designer Drugs (DREADD) into mPFC. Additionally, rats will be implanted with bilateral microinjection cannulae targeting nucleus accumbens core (AcbC) for infusion of the retrograde tracer Fluorogold prior to terminal test sessions in order to examine changes in cellular activity in mPFC to AcbC projections. Using these strategies, studies in Aim 1 will determine the functional role of the mPFC in modulating sensitivity to the N+A compound drug cue and each component alone following Gi-receptor activation (via Clozapine-N-Oxide (CNO) injection). We hypothesize that Gi-activation in mPFC will decrease sensitivity to a N+A cue and that cellular activity (measured by c-Fos) between mPFC and AcbC will be significantly decreased following CNO treatment. These two findings together would suggest a role for mPFC in modulating sensitivity to a N+A compound interoceptive cue. The studies in Aim 2, will assess the role of mPFC in extinguishing a compound N+A interoceptive cue. For these experiments, rats will undergo the same procedures outlined above; however upon reaching training criteria, they will begin non-reinforced N+A extinction sessions. For the sessions, rats will receive CNO or saline during extinction and then be assessed for reinstatement of sensitivity to the compound cue and its components. We hypothesize that silencing mPFC during extinction training will blunt the effects of extinction compared to rats treated with saline. As such, we hypothesize that rats treated with CNO during extinction will show reinstatement when re-exposed to the compound cue whereas saline treated rats will not. In contrast to Aim 1, we hypothesize that rats treated with CNO during extinction will show greater cellular activity (as measured by c-Fos) following reinstatement testing compared to saline. Taken together, the innovative and novel studies in this application will demonstrate a role for mPFC in both the expression of sensitivity and reinstatement of sensitivity (following a period of extinction) to a N+A compound interoceptive cue.